The present invention relates to a packed bed bioreactor and more particularly to a bioreactor which utilizes radial flow of the reaction medium across the packed bed.
In recent years, the use of solid support matrices for homogeneous and heterogeneous catalysis has become quite wide spread, as it facilitates control of the reaction conditions and parameters. In the face of a great deal of demand for certain types of industrially and pharmaceutically significant chemicals, the utilization of individual enzymes and/or multienzyme systems has also expanded to a wide variety of fields. The recent developments in biochemistry and biotechnology, the clarification of the mechanisms of enzyme reactions, development of new sources for various enzymes, combined with the progress in applied microbiology and genetic engineering have markedly accelerated the utilization of enzymes for various industrial processes. As a result of these technological advances and the need for the large scale production of enzymes and enzyme systems, great efforts are being directed to alternative methods for the large scale production of enzymes, rather than the traditional, tedious, time-consuming and laborious processes of producing and extracting these enzymes from naturally occurring animal and/or microbial sources, and combating problems associated with the inactivation and/or "poisoning" of these enzymes by other components of the cell systems from which they have been extracted. Methods have been developed for the direct immobilization of whole microbial or mammalian cells on inert supports and directing reaction media through such immobilized cells, whereby the enzymes contained in the cells react with the media and produce the desired products, without contamination by other cell components. Such immobilization techniques are discussed in "Immobilized Cells In Preparation Of Fine Chemicals", I. Chibata et al., "Advances in Biotechnological Processes", I, pp 203-222, 1983, published by Alan R. Liss, Inc., 150 Fifth Avenue, New York, N.Y.
Current interest in the use of biologically derived substances as therapeutic and diagnostic agents in medicine has stimulated research into improved methods for the production of these substances. Since naturally occurring biomolecules are usually present at low levels in animal and microbial sources, and because it is logistically difficult to increase production by adding more and more animals or microbial cells to a manufacturing protocol, process scale-up in vivo is very difficult and impractical. In the case of bioengineered products, there is a need for efficient expression systems for the production of recombinant-DNA proteins. These considerations have led to the development of bacterial and mammalian cell culture systems for the large-scale production of industrially useful and therapeutically valuable biomolecules.
As a result, substantial efforts have been directed to the large scale, relatively fast growth of whole naturally occurring and/or genetically altered cells, for various industrial uses. These efforts have resulted in the development of various types of bioreactors for the growth of cells and/or for the immobilization of isolated enzymes and enzyme systems. The following U.S. patents exemplify some of the different types of bioreactors used for various applications and the problems associated with each of these reactors:
U.S. Pat. Nos. 4,220,725 issued Sept. 2, 1980 to R.A. Knazek et al; 4,279,753 issued July 21, 1981 to N.E. Nielson et al; 4,391,912 issued July 5, 1983 to K. Yoshida et al; 4,442,206 issued Apr. 10, 1984 to A.S. Michaels et al; and 4,603,109 issued July 19, 1986 to E. Lillo. The U.S. Pat. No. 4,603,109 issued to Lillo sets forth the a discussion of the various bioreactor systems including the vat-type, the packed column-type, the porous membrane-type and a porous ceramic matrix-type reactors, wherein the reaction solution in introduced into an annular bore through a ceramic matrix member and the product solution is collected from the outside of the ceramic matrix member.
Various reaction schemes in which bioreactors have been utilized may be broadly classified into six major categories schematically represented as follows:
______________________________________ 1. Catalytic Reactants column packed Product Reactor with catalyst 2. Enzyme Substrate enzyme immobilized Bioreactor - I on matrix 3. Enzyme Substrate cells containing Product Bioreactor - II enzyme immo- bilized on matrix 4. Cell Culture Nutrients non-adherent Product + Bioreactor + 02 cells immobilized Waste by cross-linking on matrix 5. Cell Culture Nutrients adherent cells Product + Bioreactor - II + 02 growing on matrix Waste 6. Extra Corporeal Plasma absorption purified Shunt or other or affinity plasma or fluid from bed fluid patient returned to patient ______________________________________
For cell culture, represented in reactions 4 and 5 above, there are two basic cell types that are used in mammalian cell bioreactors--suspension cells and anchorage-dependent cells. Suspension cell lines can often be grown using modifications of classical technology, such as the stirred-tank reactor, originally developed for the growth of bacterial and yeast cells in the fermentation industry. For certain applications, these suspension cells may also be conveniently cross-linked to a solid matrix or trapped inside a suitable "holding" molecule, rather than grow them in a suspension mode.
The culture technology for anchorage-dependent or adherent cells is more sophisticated. Anchorage-dependent cells produce many substances of commercial interest but are much more difficult to grow than suspension cells. They require a solid support to which they can attach before any growth or division can occur. Their requirement of cell attachment results in some handling difficulties. They ar also generally more demanding in their nutritional requirements than are suspension cells. As a result, a number of new techniques are being developed for the large-scale production of anchorage-dependent cells, with varying degrees of success., depending on the particular cell line used. Each of these techniques, however, suffers from significant drawbacks. Therefore, there exists a great need for improved devices for the large-scale growth of anchorage-dependent cells which increase their viability and ease of handling.
Conventional large-scale production of anchorage-dependent or adherent cells uses glass of plastic roller bottles, derivatives of the flasks used in laboratory-scale research. But, roller bottles have a relatively low surface area for cell growth, are unwieldy to use in large numbers or sizes and are relatively labor-intensive. In an effort to increase the surface area, methods have been devised whereby glass spheres (diameter of 2-3 mm) are packed in a column and inoculated with the cell culture. Fluid pumps circulate nutrients from an external medium reservoir. Oxygen tension and pH need to be carefully controlled. The nutrient medium flow in this type of a glass bead bioreactor is generally from the bottom of the column, through the beads and cells, to the top of the column, where it is drawn off and recirculated. This type of flow through the length of the entire chamber can be termed longitudinal forced flow. It results in a polarized column where the culture medium encountered by the cells at the top has been partially depleted of nutrients and has a higher concentration of waste products. Furthermore, scaling up this type of column is fraught with difficulties associated with the necessity for cleaning the beads with strong acid before each use, the possibility of the increased weight of the glass beads damaging the cells, and the logistical problems caused by the shear number of beads needed for large sized columns.
A substantial advance in high-yield culture of anchorage-dependent cells has been achieved with the introduction of microcarrier heads, small spheres of dextran, agar, gelatin, polystyrene or polyacrylamide, on whose surfaces, cells can attach and grow. The microcarriers are placed in a tank-type culture vessel with the culture or growth medium and kept in suspension with gentle stirring. However, even this advanced technique presents certain procedural problems. The necessity for suspension and mixing creates handling problems. The cells are subjected to mechanical stress which might result in cell rupture. There is also a problem associated with the transfer of oxygen that is characteristic of stirred-tank reactors. In order to prevent settling of the cells out of suspension, the microcarrier density must be severely limited.
Another alternative prior art method for the large-scale culture of cells is the use of hollow fiber cartridges. Each cartridge is composed of many long, narrow polysulfone, polypropylene or polyester fibers running in parallel. The fibers are embedded at both ends in a cylindrical housing, and the device is enclosed within a plastic shell. The cells are housed on the outside of the fibers and culture medium flows longitudinally through the bores of the hollow fibers. Hydrostatic pressure differences cause some of the medium to penetrate the fiber walls and bathe the cells, which have been deposited outside the fibers in the extracapillary space. Eventually, this secondary nutrient flow returns to the lumena of the fibers and exits the cartridge.
There are, however, a number of drawbacks to this method also. The longitudinal flow of the medium results in a pressure drop from one end to the other end of the cartridge. This pressure drop produces severe gradients in the distribution of nutrients and waste products such that the culture growth is not uniform throughout the cartridge. In addition, limited or restricted circulation in the extracapillary space leads to the formation of microenvironments and anoxic pockets of nonviable cells. Finally, occlusion of the fibers by cell growth can interfere with the mass transfer of nutrients and oxygen to the cell colonies. These problems are enhanced by and during process scale-up; the bigger the cartridges, the greater is the influence of the pressure drop, anoxic pockets and microenvironments. Rather than increase fiber unit size, it appears more feasible to place numerous small cartridges in a parallel configuration.
A relatively new development in culture technology is the use of a honeycombed ceramic matrix for the cultivation of anchorage-dependent cells. The ceramic is manufactured in the form of a cylindrical cartridge and contains numerous square channels that run in parallel throughout the length of the device. Medium flow is longitudinal and in direct contact with the cells that have adhered to the ceramic. The longitudinal flow of this unit can also set up nutrient gradients similar to those noted earlier with the glass bead and hollow fiber reactors. The major problem in using and evaluating the ceramic matrix as a culture chamber is that it is only available as part of an expensive bioreactor system and has not been widely distributed or available for widespread use.
Commercially valuable biomolecules derived from cultured cells, or enzymes are usually secreted in minute quantities. Therefore, the highest possible cell densities or concentrations and largest-scale cultures are needed to achieve economical and large quantity production of these substances of interest. Since the role of cultured cells and enzymes in the production of valuable molecules is likely to increase in the near future, efficient means for their production, propagation and utilization is of paramount importance. In the case of anchorage-dependent cells, new culture methods and devices which would help in the elimination of the problems of low growth-surface area, low cell yield, unequal distribution of nutrients, and inefficient scale-up is essential. In the case of enzymes, methods and devices which maximize their catalytic efficiency and viability are highly desirable. Thus a need exists for a device and method which can be easily adapted for use as a chemical reactor, a bioreactor, a cell culture chamber or reactor, or as an extra corporeal shunt and which also successfully addresses, minimizes and/or eliminates the problems of the prior art devices.
Therefore, it is an object of this invention to provide an improved bioreactor which is easily adaptable for reactions involving cells or cell components or inorganic or organic catalysts including enzymes or enzyme systems.
A further object of the invention is to provide a cell culture growth chamber for anchorage-dependent cells.
Another object of the invention is to provide a cell culture growth chamber for animal, plant or microbial cells.
Still another object of the invention is to provide a cell culture growth chamber for cells in suspension or nonanchorage-dependent cells.
Yet another object of the invention is to provide a bioreactor which utilizes a radial or horizontal flow of culture or reaction medium across a packed bed of support material.
Still another object of the invention is to provide a cell growth chamber which eliminates the problems of a low growth surface area, low cell yield and unequal distribution of nutrients to the cell culture.
Another object of the invention is to provide a cell culture growth chamber that is capable of achieving high cell densities and which can be easily scaled up, while providing uniform distribution of the culture medium.
Another object is to provide an enzyme bioreactor which maximizes the enzyme efficiency and the life time of the enzyme.
Yet a further object of the invention is to provide a growth chamber for large-scale preparation of anchorage-dependent cell culture.
Another object of the invention is to provide a bioreactor having the capabilities of commercial application in the production of medically relevant, enzyme or cell-culture-derived molecules such as anti-tumor factors, hormones, therapeutic enzymes, viral antigens, interferons and other substances.
Yet another object of the invention is to provide a bioreactor which utilizes a radial or horizontal flow of culture or reaction medium across a packed bed of microcarriers or other support matrices with enzymes or enzyme systems attached thereto for reaction with selected substrates of choice.
Another object of the invention is to provide a bioreactor having the capabilities of commercial application in the production of commercially viable, cell-culture-dependent or chemically catalyzed or enzyme-catalyzed reaction products in the food, agriculture, oil and other industries.
Additional objects, advantages and novel features of the invention will be set forth in part in the description which follows, and in part will become apparent to those skilled in the art upon examination of the following or may be learned by practice of the invention. The objects and advantages of the invention may be realized and attained by means of the instrumentalities and combinations particularly pointed out in the appended claims.